Ph-sensitive polymer

ABSTRACT

The invention relates to a pH-sensitive polymer which is a (meth)acrylate copolymer composed of 20 to 65% by weight acrylic and/or methacrylic acid units and 80 to 35% by weight units of C 1 - to C 18 -alkyl esters of (meth)acrylic acid, characterized in that it has a molecular weight in the range from 1 000 to 50 000 g/mol, and brings about at least 60% haemolysis at pH 5.5, and less than 5% haemolysis at pH 7.4, in a concentration of 150 μg/ml in a cytotoxicity test with human red blood cells. The invention further relates to the use of the pH-sensitive polymer as carrier for pharmaceutically effective biomolecules or active pharmaceutical ingredients and as ingredient of corresponding dosage forms.

The invention relates to a pH-sensitive polymer which has cytotoxic ormembranolytic properties at pH values below pH 6.5 and which can be usedas carrier for natural or synthetic biomolecules or activepharmaceutical ingredients.

PRIOR ART

Polymers which respond to stimuli have increased in importance in recentyears. Corresponding polymers display modified properties after exposureto a chemical or physical influence such as, for example, to mentiononly a few, temperature, solvent or pH. There is particular interest inthis connection in pH-sensitive polymers. Thus, for example, carboxylgroup-containing polymers which form hydrophilic coil structures at highpH may be converted at low pH values into hydrophobic globule structures(see, for example, (1)) .

Research is focused on pH-sensitive polymers in connection with theadministration of medicinal substances. Many physiological andpathological processes such as endocytosis, tumour growth andinflammations are associated with a change in pH conditions.. Examplesof pH-sensitive polymers being investigated in connection with theadministration of medicinal substances are, for example, α-alkylacrylicacids such as poly(2-ethylacrylic acid) and poly(propylacrylic acid)(see 2), poly(amido amines) (see 3, 4) and poly(ethylenimine),poly(L-lysine isophthalamide) (see (5)). The conformational changesinduced by pH shifts influence the interactions of polymer and cellmembranes in such a way that destabilization may occur. It is possibleto employ, complex or conjugate pH-sensitive polymers as means fortransporting with a large number of natural or synthetic biomolecules.They can be complexed or conjugated with lipids (see 6, 7, 8, 14),proteins and peptides (see 9, 10), DNA (4, 11), immunotoxins (12),antibodies (13) and/or active ingredients (3).

LIST OF THE LITERATURE CITED ABOVE

-   1. M. Watanabe, Y. Kosaka, K. Sanui, N. Ogata, K. Ogushi, and T.    Yoden. On the mechanism of polyelectrolyte-induced structural    reorganization in thin molecular films. Macromolecules, 20: 454-456    (1987).-   2. N. Murthy, J. R. Robichaud, D. A. Tirrell, P. S. Stayton, and S.    Hoffmann. The design and synthesis of polymers for eukaryotic    membrane disruption. J. Controlled Release, 61: 137-143 (1999).-   3. R. Duncan, P. Ferruti, D. Sgouras, A. Tuboku-Metzger, E. Ranucci,    and F. Bignotti. A polymer-Triton X-100 conjugate capable of    pH-dependent red blood cell lysis: A model system illustrating the    possibility of drug delivery within acidic intracellular    compartments. J. Drug Targeting, 2: 341-347 (1994).-   4. S. C. Richardson, N. G. Pattrick, Y. K. Stella Man, P. Ferruti,    and R. Duncan. Poly(amidoamine)s as potential nonviral vectors:    ability to form interpolyelectrolyte complexes and to mediate    transfection in vitro. Biomacromolecules, in press (2001).-   5. M. E. Eccleston, M. Kuiper, F. M. Gilchrist, and N. K. H. Slater.    pH-responsive pseudo-peptides for cell membrane disruption. J.    Controlled Release, 69: 297-307 (2000).-   6. T. Chen, L. S. Choi, S. Einstein, M. A. Klippenstein, P.    Scherrer, and P. R. Cullis. Proton-induced permeability and fusion    of large unilamellar vesicles by covalently conjugated    poly(2-ethylacrylic acid). J. Liposome Res., 9: 387-405 (1999).-   7. J. L. Thomas and D. A. Tirrell. Polyelectrolyte-sensitized    phospholipid vesicles. Acc. Chem. Res. 35: 336-342 (1992).-   8. X. Guo and F. C. Szoka Jr. Steric stabilization of fusogenic    liposomes by a low-pH sensitive PEG-diortho ester-lipid conjugate.    Bioconjugate Chem., 12: 291-300 (2001).-   9. C. A. Lackey, N. Murthy, O. W. Press, D. A. Tirrell, A. S.    Hoffmann, and P. S. Stayton. Hemolytic activity of pH-responsive    polymer-streptavidin bioconjugates. Bioconjugate Chem., 10: 401-405    (1999).-   10. V. Bulmus, Z. Ding, C. J. Long, P. S. Stayton, and A. S.    Hoffman. Site-specific polymer-streptavidin bioconjugate for    pH-controlled binding and triggered release of biotin. Bioconjugate    Chem., 11: 78-83 (2000).-   11. P. S. Stayton, N. Murthy, C. Lackey, C. Cheung, R. To, O. Press,    and A. S. Hoffman. Bioinspired polymers designed to enhance    intracellular delivery of biotherapeutics. Proceed. Intern. Symp.    Control. Rel. Bioact. Mater., 27: 7330 (2000).-   12. C. A. Lackey, N. Murthy, P. S. Stayton, O. W. Press, A. S.    Hoffman, and D. A. Tirrell. Enhancement of endosomal release and    toxic activity of ricin A chain by a pH-sensitive polymer. Proceed.    Intern. Symp. Control. Rel. Bioact. Mater., 26: 815-816 (1999).-   13. C. A. Lackey, O. W. Press, A. S. Hoffman, and P. S. Stayton.    pH-sensitive polymer-protein complexes for control of intracellular    trafficking of biomolecular therapeutics. Polym. Mater. Sci. Eng.,    84 (2001).-   14. K. M. Eum, K. H. Langley, and D. A. Tirrell. Quasi-elastic and    electrophoretic light scattering studies of the reorganization of    dioleoylphosphatidylcholine vesicle membranes by poly(2-ethylacrylic    acid). Macromolecules, 22: 2755-2760. (1989).

WO 97/09068 describes interactive molecular conjugates. These are inparticular molecules which respond to a stimulus and which have theability to bind to a cellular target region, the stimulus in turninfluencing the binding ability. The stimulus may be given by thetemperature, pH, particular ions or ionic strengths, electric fields orsolvents.

A molecule which responds to a stimulus may be, for example, apH-sensitive polymer which is combined with a molecule-which binds to aligand, e.g. an antigen, an antibody or an active pharmaceuticalingredient. The molecular conjugate is able to respond to alteredenvironmental conditions, e.g. to alteration of the pH from values abovepH 7.0 in the region of extracellular body fluids, e.g. in the bloodstream, to values below pH 6.5, which is associated with uptake intoliving cells, e.g. by endocytosis.

The polymers generally mentioned are based on vinyl-type monomers whichhave undergone free-radical polymerization and have molecular weights inthe range from 1 000 to 30 000. Polymers with reactive side groups, e.g.—OH, —COOH or, preferably, —NH₂, are suggested for coupling proteins orpeptides.

(Meth)acrylate copolymers composed of methacrylic acid units andcomonomers such as C₁- to C₁₂-alkyl esters of (meth)acrylic acid areknown in principle and are used in particular as coating agents andbinders for drug forms.

Known examples are copolymers composed of 50% by weight methacrylic acidand 50% by weight methyl methacrylate (EUDRAGIT® L), of 50% by weightmethacrylic acid and 50% by weight ethyl acrylate (EUDRAGIT® L100-55),of 30% by weight methacrylic acid and 70% by weight methyl methactylate(EUDRAGIT® S). The commercially available copolymers have molecularweights in the region of about 200 000 g/mol.

US 2001/0007666 A1 describes a composition used to increase thetransport or release of substances through cell membranes, betweencells, cell compartments or lipid membranes. The composition consists ofa membrane transport agent which may inter alia be a pH-sensitivepolymer, and physical means to increase the efficiency of the membranetransport agent, e.g. ultrasonic treatment. Particularly suitablepH-sensitive polymers are carboxyl group-containing polymers, e.g. thosecontaining more than 50% monomer residues with carboxyl is groups. Aspecific example mentioned is poly(2-ethylacrylic acid) with an averagemolecular weight of 62 000. 50:50 copolymers of acrylic acid with ethyl,propyl and butyl acrylates are also described. Preparation is stated tobe by bulk polymerization in the presence of an AIBN initiator.Molecular weight regulators are not mentioned. Since the copolymers aremoreover purified by ether precipitation, the molecular weights must beassumed to be high. Even in low concentrations of a few μg, thecopolymers lyse erythocytes extensively at pH 5.5 and to a smallerextent at pH 7.4 at least.

PROBLEM AND SOLUTION

The introduction of biomolecules or active pharmaceutical ingredientsinto the cytoplasm and from there possibly into the cell nucleus viaendosomes requires membrane-destabilizing (membrane-destroying) agentswhich prevent the substances traffic to (entering) the lysosomes, wherethey may be chemically degraded or inactivated. The polymers of interesttherefore lead to destabilizing (destruction) of cell membranes atslightly acidic pH values around pH 6.5 or below, as prevail in theendosomes, but are substantially devoid of (have no) membranolyticeffect at physiological pH values around pH 7.4, as occur outside thecells. The acrylic acid/alkyl acrylate copolymers of US 2001/0007666 A1have the disadvantage that they may lead even in extremely lowconcentrations to cytolysis. This makes dosage thereof critical. Anadditional disadvantage is that some haemolysis may occur even at pH7.4, so that the described copolymers are overall difficult to controland, if they are used in extremely small amounts, only low loading withbiomolecules or active pharmaceutical ingredients is made possible. Anadditional disadvantage is that following parenteral administration,they might accumulate in the body. When the molecular weight of apolymer is too high, for example 62,000, excretion by glomerularfiltration is prevented.

In connection with the development of drug forms intended to displaytheir effects specifically in particular cell types, therefore, there isa continuing need for suitable polymeric carrier molecules. Theintention was to develop pH-sensitive polymers which display cytotoxicor membranolytic properties only in high concentrations, or not at all,in the region of pH 7.0 or slightly higher, but have cytotoxic, orhaemolytic or membranolytic effects even in low concentration in vivobelow pH 6.5. The polymers are intended to be suitable as carriers(complexes and conjugates) for natural or synthetic biomolecules oractive pharmaceutical ingredients which are to be released inside cellsvia uptake into endosomes and subsequent destabilization or lysisthereof.

Furthermore in the parenteral application of substances the problem ofelimination has to be regarded in order to avoid an accumulation in thehuman or animal body and therefore toxic side effects.

Therefore pH-sensitive polymers should be provided which can be easilyapplied at concentrations in the range of for example 20 to 150 μg/mland which are well chargeable and which therefore are suitable ascarriers (complexes and conjugates) for bio-molecules, of natural orsynthetic origin. They shall show good haemolytic properties in thisrange of concentration and in the range of pH 5.5 and below pH 6.5,while there shall be no membranolytic (haemolytic) effect at pH 7.4.Furthermore they shall not be effective against macrophage cell types bybeing toxic or being inhibiting. The polymers shall be eliminated wellvia the kidney and therefore be suitable also for parenteralapplications.

The problem is solved by a

-   pH-sensitive polymer which is a (meth)acrylic copolymer composed of    -   20 to 65%, 25 to 65% by weight methacrylic acid units and    -   80 to 35%, 75 to 35 by weight units of C₁- to C₁₈-alkyl esters        of (meth)acrylic acid,        characterized in that-   it has a molecular weight in the range from 1 000 to 50 000 g/mol,-   and brings about at least 60% haemolysis at pH 5.5, and less than 5%    haemolysis at pH 7.4, in a concentration of 150 μg/ml in a    cytotoxicity test with human red blood cells.

Because poly(meth)acrylates—in contrast to other pharmaceutically usedpolymers—are not biologically degradable, the elimination must beeffected by glomaerulous filtration via the kidney. However this processis restricted in respect to the molecular weight. An upper limit formolecules that can be secreted via the kidney is adopted with 50.000g/mole (dalton). Surprisingly polymers with low molecular weightsaccording to the present invention show higher haemolytic activitiesthan those with higher molecular weights.

IMPLEMENTATION OF THE INVENTION

(Meth)acrylate copolymers

The invention relates to a

pH-sensitive polymer, in particular with membranolytic, haemolytic orcytotoxic properties at pH values below pH 6.5, which is a(meth)acrylate copolymer composed of

20 to 65% by weight methacrylic acid units and 80 to 35% by weight unitsof C₁- to C₁₈-alkyl esters of (meth)acrylic acid,

C₁- to C₁₈-alkyl esters of (meth)acrylic acid, in particular linear orbranched C₁- to C₁₈-alkyl esters of (meth)acrylic acid, are, forexample:

methyl acrylate, ethyl acrylate, vinyl acrylate, propyl acrylate, butylacrylate, hexyl acrylate, octyl acrylate, decyl acrylate, dodecylacrylate, myristyl acrylate, lauryl acrylate, cetyl acrylate, stearylacrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate,butyl methacrylate, isobutyl methacrylate, hexyl methacrylate,2-ethylhexyl-(meth)acrylate, phenyl methacrylate, octyl methacrylate,decyl methacrylate, dodecyl methacrylate, myristyl methacrylate, laurylmethacrylate, cetyl methacrylate, stearyl methacrylate

The ester components may be branched or cyclic.

Preference is given to the C₁- to C₈-alkyl esters of acrylic ormethacrylic acid, in particular methyl methacrylate, ethyl methacrylate,butyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethylacrylate, butyl acrylate and 2-ethylhexyl acrylate.

As a rule, the stated contents add up to 100% by weight. However, italso possible, without this necessarily leading to an impairment oralteration in the essential properties, for small amounts in the rangefrom 0 to 10, e.g. 0.1 to 5, or not more than 2.5, % by weight of othervinylically copolymerizable monomers, which are not necessarily(meth)acrylates, to be present, e.g. butyl acrylate, butyl methacrylate,methyl acrylate, hydroxyethyl methacrylate, hydroxyethyl acrylate,methacrylamide or styrene. Crosslinking monomers are not present as arule.

(Meth)acrylate copolymer variants

Suitable and preferred for the purposes of the invention are(meth)acrylate copolymers composed of:

-   -   40 to 60, in particular 45 to 55, e.g. 50, % by weight        methacrylic acid units and 60 to 40, in particular 55 to 45,        e.g. 50, % by weight ethyl acrylate units (EUDRAGIT® L100-55        type).    -   40 to 60, in particular 45 to 55, e.g. 50, % by weight        methacrylic acid units and 60 to 30, in particular 45 to 35,        e.g. 50, % by weight ethyl acrylate units, and 2 to 20% by        weight, e.g. 10% by weight, butyl methacrylate.    -   40 to 60, in particular 45 to 55, e.g. 50, % by weight        methacrylic acid units and 60 to 40, in particular 55 to 45,        e.g. 50, % by weight ethyl acrylate units, and 0.1 to 2% by        weight of a C₈- to C₁₆, preferably C₈-C₁₂-alkyl ester of acrylic        or methacrylic acid, preferably dodecyl methacrylate. The        copolymer can preferably be prepared in the presence of 5 to 15%        by weight dodecyl mercaptan or 2 to 10% by weight 2-ethylhexyl        thioglycolate and varies accordingly in its properties.    -   20 to 40, in particular 25 to 35, e.g. 30, % by weight        methacrylic acid units, 25 to 45, in particular 30 to 40, e.g.        35, % by weight methyl methacrylate units, 25 to 45, in        particular 30 to 40, e.g. 35, % by weight ethyl acrylate units.

As a rule, the stated contents add up to 100% by weight. However, italso possible, without this necessarily leading to an impairment oralteration in the essential properties, for small amounts in the rangefrom 0 to 10, e.g. 0.1 to 5, or not more than 2.5, % by weight of othervinylically copolymerizable monomers, which are not necessarily(meth)acrylates, to be present. For example, butyl acrylate, butylmethacrylate, methyl acrylate, hydroxyethyl methacrylate, hydroxyethylacrylate, methacrylamide or styrene may be mentioned.

Molecular weight

The molecular weight can be determined for example by viscometry or gelexclusion chromatography (GPC). Viscometric values (limiting viscositynumber) can be determined in chloroform or in DMF (dimethylformamide) at23° C. and should preferably be in the range from 1 to 25, preferably 10to 20, n_(spec/c) (cm³/g). Viscosity numbers can be measured for exampleas specified in ISO 1628-6. The molecular weights according to thisinvention should be determined by viscosimetry according to DIN 1628-6,modified with DMF as a solvent. Viscosity numbers are correlated to themolecular weight (weight average, M_(w)) using poly methylmethacrylate(PMMA)-standards. The molecular weight (weight average) of the(meth)acrylate copolymer is in the range from 1 000 to 50 000 g/mol,preferably in the range from 5 000 to 40 000 g/mol, in particular in therange from 10 000 to 30 000 or most preferably in the range of 15 000 to25 000.

The following table may be used as a guideline to calculate molecularweights from viscosity values (VZ) according to the general formulaVZ=4,976 10⁻³*M_(w) ^(0,796): Viscosity number (VZ) Molecular weight(M_(w)) 1 780 3 3110 5 5910 10 14110 15 23490 20 33720 30 56110 50106600

Haemolytic effect

If the haemolytic activity (haemolysis) exceeds 5% there is said to be acytotoxic effect. The haemolytic activity should be less than 5% at pH7.4 and be high and be, for example, at least 30, at least 40, at least50 or at least 60, % at pH 5.5. It is beneficial if this effect isreached with readily dosed copolymer concentrations of 20 to 300,preferably 50 to 250, in particular of 100 to 200, μg of copolymer/mlbased on an erythrocyte concentration of 1×10⁸ RBC/ml.

Most preferred are copolymer concentrations of 10 to 150, preferred from15 to 110, in particular from 20 to 80 μg copolymer/ml.

In a cytotoxicity test with human red blood cells, the (meth)acryliccopolymer at a concentration of 150 μg/ml brings about at least 60%,preferably at least 80%, haemolysis at pH 5.5, and less than 5%,preferably less than 2.5, particularly preferably less than 1, %haemolysis at pH 7.4.

The cytotoxicity test with human red blood cells (erythrocytes) can becarried out by a method based on that of Murthy et al.: N. Murthy, J. R.Robichaud, D. A. Tirrell, P. S. Stayton, and S. Hoffman. The design andsynthesis of polymers for eukaryotic membrane disruption. J. ControlledRelease, 61: 137-143 (1999).

This entails human red blood cells (RBC) obtained from fresh blood beingseparated by centrifugation in the presence of K3 EDTA. The cells are inthis case sedimented at 200 g and 4° C. for 5 min and subsequentlywashed three times by renewed centrifugation and taking up inphosphate-buffered saline (PBS buffer, 34 mM, pH 7.4, 0.9% NaClweight/volume (75 mM)). The cell count in the resulting suspension canbe determined using a haemocytometer.

The haemolysis test is carried out by adding the human red blood cells(RBC) in the particular medium to a copolymer suspension at a cellconcentration of 1×10⁸ RBC/ml. The mixture is incubated at 37° C. for 20min.

Cytotoxic effect on macrophage-type cells

In contrast to red blood cells, where the cytolysis presumably takesplace mainly through an interaction of the copolymers with the outercell membrane, the macrophage-like cell types are capable of activeuptake of the copolymers, so that it must be assumed that otherinteractions may cause cytolysis in this case. The preferred(meth)acrylate copolymers are those showing low or zero cytolysis ortoxicity in the MTT test or in the LDH test (lactate dehydrogenase test)with the mouse macrophage-like cell type J774A.1 (see Example 5). Thecell line J774A.1 is available, for example, from the public collectionof strains ATCC (America Type Culture Collection, Manassas, Va. 20108)under the No. TIB-67 (J774A.1 mouse cells; immortalized macrophage-likecells (not tumoral); see also: Ralph P et al. Lysozyme synthesis byestablished human and murine histiocytic lymphoma cell,. lines. J. Exp.Med. 143: 1528-1533, 1976 PubMed: 76192838.

MTT test (inhibition of cells of a macrophage-type cell line):

The calorimetric test detects living cells which reduce the yellow MTTdye (MTT=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)to a dark blue formazan product. The test is suitable for detectingcytotoxicity, cell proliferation and cell activation (see, for example,T. Mosmann. Rapid calorimetric assay for cellular growth and survival:Application to proliferation and cytotoxicity assays, J. Immunol. Meth.65:55-63 (1983)).

-   -   The extent of killing of macrophage cells is indicated by the        LDH released (N C. Phillips, L. Gagné, N. Ivanoff, g.        Riveau. (1996) Vaccine 14(9), 898-904. The lactate dehydrogenase        test for measuring the released LDH (see P. G. Cabaud and F.        Wroblewski in Am J Clin Pathol (1958) 30, 234-236) can be        carried out with commercially available test kits, e.g. Sigma        kit (procedure No. 500).

Toxicity induced by the copolymers towards macrophage-like cells shouldbe as low as possible at physiological (neutral) pH.

Preferred (meth)acrylate copolymers are therefore those which, at aconcentration in the region of 0.03125 mg/ml, bring about in the MTTtest with the macrophage-like cell type J774A.1 (ATCC TIB-67) aperventage-value of cell survival of at least 25%, preferably at least60%, based on a 100% survival rate in the control experiment.

Preferred (meth)acrylate copolymers are therefore those which, at aconcentration in the region of 0.03125 mg/ml, bring about in the LDHtest with the macrophage-like cell type J774A.1 (ATCC TIB-67) a LDHrelease-value of not more than 40%, preferably not more than 20%, basedon a 100% cytolysis (toxicity) in the control experiment.

Preparation of the pH-sensitive polymers or (meth)acrylate copolymers

The pH-sensitive polymers are prepared by free-radical polymerization ofthe monomers in the presence of polymerization initiators and molecularweight regulators by block, bead or emulsion polymerization anddischarge of the polymer. Other preparation methods which are suitablein principle are also group transfer polymerization (GTP) or atomtransfer radical polymerization (ATRP) (see, for example, Matyjaszewski,K. et al., Chem. Rev. 2001, 101, 2921-2990). The resulting polymerstructures are random copolymers or block copolymers.

Preference is given to emulsion polymerization in the presence of 2 to15% by weight molecular weight regulators, an emulsifier content in therange from 0.1 to 2% by weight, an amount of polymerization initiator inthe range from 0.02 to 0.4% by weight and at temperatures from 65 to 90°C. Preference is given to an emulsifier mixture preferably composed ofsodium lauryl sulphate, e.g. 0.1 to 0.5% by weight, and polyoxyethylene20 sorbitan monooleate, e.g. 0.4 to 1.5% by weight. Particularlysuitable initiators are sodium peroxodisulphate or ammoniumperoxodisulphate. It is possible in this way to prepare, for example, adispersion with a solids content of 20 to 40% by weight, and thecopolymer can be isolate, preferably by spray drying or by coagulationand expelling the water in an extruder. The polymer is then dissolved,preferably in an organic solvent, purified, preferably by multipledialysis against water, and dried, preferably freeze dried.

Examples of polymerization initiators which may be mentioned are: azocompounds such as 2,2′-azobis(isobutyronitrile) or2,2′-azobis(2,4-dimethylvaleronitrile), redox systems such as, forexample, the combination of tertiary amines with peroxides or,preferably, peroxides (concerning this, see, for example, H.Rauch-Puntigam, Th. Völker, “Acryl- und Methacrylverbindungen”,Springer, Heidelberg, 1967 or Kirk-Othmer, Encyclopedia of ChemicalTechnology, Vol. 1, pages 386 et seq., J. Wiley, New York, 1978).Examples of suitable peroxide polymerization initiators are dilauroylperoxide, tert-butyl peroctoate, tert-butyl perisononanoate,dicyclohexyl peroxydicarbonate, dibenzoyl peroxide or2,2-bis(tert-butylperoxy)butane.

It is also possible and preferred for the polymerization to be carriedout with a mixture of various polymerization initiators differing inhalf-life, for example, dilauroyl peroxide and2,2-bis(tert-butylperoxy)butane, in order to keep the flow of freeradicals constant during the polymerization and at differentpolymerization temperatures. The amounts of polymerization initiatoremployed are generally from 0.01 to a maximum of 1% by weight based onthe monomer mixture.

The molecular weights of the copolymers (CP) are adjusted bypolymerizing the monomer mixture in the presence of molecular weightregulators, such as, in particular, of the mercaptans known for thispurpose, such as, for example, n-butyl mercaptan, n-dodecyl mercaptan,2-mercaptoethanol or 2-ethylhexyl thioglycolate, generally employing themolecular weight regulators in amounts of 0.05 to 15% by weight based onthe monomer mixture, preferably in amounts of 0.1 to 10% by weight andparticularly preferably in amounts of 2 to 12% by weight of the monomermixture (cf., for example, H. Rauch-Puntigam, Th. Völker, “Acryl- undMethacrylverbindunigen”, Springer, Heidelberg, 1967; Houben-Weyl,Methoden der organischen Chemie, Vol. XIV/1, page 66, Georg Thieme,Stuttgart, 1961 or Kirk-Othmer, Encyclopedia of Chemical Technology,Vol. 1, pages 296 et seq., J. Wiley, New York, 1978). The molecularweight regulator preferably employed is n-dodecyl mercaptan or2-ethylhexyl thioglycolate. Ethylhexyl thioglycolate has the advantagethat the hydrophobicity of the (meth)acrylate copolymer can beinfluenced since the regulator is included in the molecule at theterminus. 5 to 15% by weight dodecyl mercaptan or 2 to 10% by weight2-ethylhexyl thioglycolate are preferred amounts employed.

Conjugates/Complexes

The pH-sensitive polymer can be as intended in the form of a conjugateor complex with a pharmaceutically effective natural or syntheticbiomolecule or an active pharmaceutical ingredient. The conjugate orcomplex can be prepared reversibly or irreversibly covalently bychemical linkage or through secondary valencies via Van der Waalsforces, ionic bonds, hydrophobic linkage.

Uses

The pH-sensitive polymers can be used as carriers, conjugates/complexesfor natural or synthetic biomolecules or active pharmaceuticalingredients which are to be released inside cells via uptake intoendosomes and subsequent destabilization or lysis thereof.

The pH-sensitive polymers can be used for complexation or as conjugatedwith lipids, proteins, peptides, nucleic acids (DNA ad RNA), inparticular oligonucleotides, antisense DNA or antisense RNA, plasmidDNA, nucleotides, hormones, toxins, immunotoxins, antibodies orfragments thereof or active pharmaceutical ingredients. The complexationor conjugate formation can take place reversibly or irreversiblycovalently by chemical linkage or through secondary valencies via Vander Waals forces, ionic bonds, hydrophobic linkage. The resultingcomplex can be used as active ingredient for producing a drug form, inparticular a dermal, transdermal, parenteral, nasal, pulmonary, vaginalor oral drug form.

The pH-sensitive polymers and the conjugates may, where appropriate, bea constituent of microparticles, nanoparticles, liposomes, emulsionsand/or lipid vesicles.

The said use as carrier, conjugate/complex is possible and preferred foractive pharmaceutical ingredients from the active ingredient classes ofanalgesics, antiallergics, antirheumatics, antibiotics, antiinfectives,antiparkinson agents, antipsoriatics, antitumour agents,dermatologicals, gout remedies, immunoregulators, gastrointestinalagents, neurotropic agents, ophthalmologicals, cytostatics.

Possible disorders:

cancer, infections (including HIV), cardiovascular disorders (e.g.arteriosclerosis), arthritis, neurodegenerative disorders (Parkinsonism,multiple sclerosis, Alzheimer's), genetically related enzyme-deficiencydisorders, hepatitis B and C, mucoviscidosis, hypercholesteraemia,Down's syndrome, muscular dystrophy, autoimmune diseases, shingles andherpes, psoriasis, CMV retinitis, Crohn's disease, ulcerative colitis,diabetes.

The medicinal substances employed for the purpose of the invention areintended to be used on or in the human or animal body in order

-   1. to cure, alleviate, prevent or diagnose disorders, conditions,    physical damage or pathological symptoms.-   2. to reveal the condition, the status or the functions of the body    or mental states.-   3. to replace active substances or body fluids produced by the human    or animal body.-   4. to ward off, to eliminate or to render harmless pathogens,    parasites or exogenous substances, or-   5. to influence the condition, the status or the functions of the    body or mental states.

Medicinal substances in use are to be found in reference works such as,for example, the Rote Liste or the Merck Index.

Particular mention should be made of the active ingredients amphotericinB, cytosine arabonoside Adriamycin. The liposomal form of adriamycincould also be a good model since its fast release in endosomes could beused to overcome multidrug resistance. Other active ingredients ofinterest could be antisense oligonucleotides, plasmid DNA and peptides(leuprolide, calcitonin etc.).

The drug form can be used for example for the therapy of tumours,cardiovascular disorders or rheumatoid arthritis. Gene therapy ofgenetic disorders or prophylactic therapy of such disorders with the aidof the pH-sensitive polymers according to the invention as ingredient ofappropriate drug forms is likewise conceivable in future. Compared withviral systems for transferring nucleic acids into living cells, nonviralsystems have the general advantage that they can be prepared moreeasily, no replication of the vector is involved, and that the risk ofimmunological reactions is lower.

Modifications of the properties

a) Influencing the conformation by binding other molecules

The properties, e.g. the toxic or membranolytic activity, of the(meth)acrylate copolymers on red blood cells and/or macrophage-typecells or the physical properties thereof can be modified by covalent orsecondary valency coupling with other molecules, in particularconformation-altering agents, e.g. dyes, in particular hydrophobic dyes,e.g. with rhodamine B. Suitable coupling methods for carboxylgroup-containing (meth)acrylate copolymers are known to the skilledperson.

It is possible to obtain for example a (meth)acrylate copolymer composedof

-   -   25 to 65% by weight methacrylic acid units and 75 to 35% by        weight units of C₁- to C₁₂-alkyl esters of (meth)acrylic acid,        which is modified or coupled with a dye, preferably with a        hydrophobic dye, in particular with rhodamine B.

Preference is given to a (meth)acrylate copolymer composed of

-   -   40 to 60% by weight methacrylic acid units and 60 to 40% by        weight ethyl acrylate units,        which is modified with a dye, preferably with an in particular        hydrophobic dye, in particular with rhodamine B.

Preference is given to a theoretically determined coupling ratio inwhich 5 to 100%, preferably 5 to 20%, of the copolymer molecules arecoupled with a dye molecule.

The coupling has the advantage for example that the solubilitycharacteristics can be modified. It is possible, for example, to achievea steep rise in insolubility in the region below pH 6.5, e.g. in therange from pH 6.0 to pH 5.0 (see Example 6).

b) Production of complexes by intermolecular crosslinking

The carboxyl groups of the (meth)acrylate copolymers are chemicallyreactive and are suitable for the modification with the aim of producingcomplexes by intermolecular crosslinking. Thus, for example, SH groupscan be introduced relatively easily into the (meth)acrylate copolymersby chemical modification with NH₂—CH₂—CH₂—SH.

It is likewise easy to introduce SH groups into DNA and RNA, inparticular oligonucleotides, antisense DNA or antisense RNA, via the 5′-and 3′-terminal OH groups of nucleic acids by means of COOH—CH₂—CH₂—SH.

SH-modified (meth)acrylate copolymers and SH-modified nucleic acids canbe crosslinked by forming S—S bridges to give complexes. The increase inthe molecular weight and the reduced solubility makes the complexesavailable in granule form in particular to phagocytotic cells, which maybe advantageous in the treatment of certain pathological states such as,for example, (auto)immune diseases.

Reduction of the disulfide bridge in vivo would release the active formof the carried molecule. The complexes could also be internalised byother cell types after grafting appropriate targeting ligands.

The polymers according to the invention are employed in combination withactive ingredients and form a drug delivery system which is particulateas a rule. It is possible for this purpose to link the active ingredientto the polymer via a biodegradable spacer. However, preference is alsogiven to conjugates/complexes with cationic low molecular weight orpolymeric substances and pharmalogically active agents. Mention shouldbe made of cationic lipids such as Lipofectin, polylysine,polyethyleneimine, polyamino(meth)acrylate or spermine and spermidinesand derivatives thereof. This complex may also be a cationic or anionicliposome. Polymer and active agent may also be incapsulated in or boundto a cationic, anionic or neutral liposome.

The complexing agents which are known per se may displayaction-enhancing effects in a variety of ways (e.g. polyethyleneimine).

Further constituents of the complexes may be hydrophilic polymers(polyethylene glycol, polyvinylpyrrolidone, polylactides,polyglycolides, polysaccharides and derivatives thereof). Thesesubstances protect the active ingredients from interactions withconstituents of the blood and prolong the circulation in the blood.

Targeting ligands, such as antibodies against cell-specific antigens canbe used for cell-specific targeting.

The particulate release systems are produced by conventional techniques,e.g. by direct complexation in solution, drying of lipid-containingsolutions and redispersion in water, where appropriate using ultrasoundor homogenization.

Ingredient of a drug form which corresponds to the usual technique ofthe intended use and, permits a safe under tolerated therapy. Thestability can be extended by freeze- or spray-drying the drug forms.

EXAMPLES

1. Copolymers of Examples 1 to 7

Copolymer A:

Copolymer of

-   -   50% by weight methacrylic acid and    -   50% by weight methyl methacrylate.

Weight average molecular weight=M_(w) about 25 000.

Copolymer B:

Copolymer of

-   -   50% by weight methacrylic acid and    -   50% by weight ethyl acrylate.

Weight average molecular weight=M_(w) about 25 000.

Copolymer C:

Copolymer of

-   -   30% by weight methacrylic acid,    -   35% by weight ethyl acrylate and    -   35% by weight methyl acrylate

Weight average molecular weight=M_(w) about 25 000.

Copolymer D:

Copolymer of

-   -   30% by weight methacrylic acid,    -   70% by weight methyl methacrylate.

Weight average molecular weight=M_(w) about 25 000.

Copolymer E (not according to the invention)

Copolymer of

-   -   10% by weight methacrylic acid,    -   45% by weight methyl methacrylate and    -   45% by weight methyl acrylate

Weight average molecular weight=M_(w) about 25 000.

Copolymer L-100 (EUDRAGIT® L, not according to the invention)

Copolymer of

-   -   50% by weight methacrylic acid and    -   50% by weight methyl methacrylate.

Weight average molecular weight=M_(w) about 100 000.

Copolymer L-100-55 (EUDRAGIT® L100-55, not according to the invention)

Copolymer of

-   -   50% by weight methacrylic acid and    -   50% by weight ethyl acrylate.

Weight average molecular weight=M_(w) about 250 000.

Copolymer S-100 (EUDRAGIT® S100, not according to the invention)

Copolymer of

-   -   30% by weight methacrylic acid and    -   70% by weight methyl methacrylate.

Weight average molecular weight=M_(w) about 100 000.

Example 1 pH Transition Ranges (Soluble to Insoluble)

The intention was to investigate, by measuring the scattered light at37° C. at pH values from 3.0 to 7.5 in phosphate buffer, the pH rangesin which the copolymers are present in insoluble form. The intensity oflight scattering increases as the polymer precipitates.

The results are shown in Table 1 below TABLE 1 pH range in MethacrylicMethyl Ethyl Methyl which the acid methacrylate acrylate acrylate Mw ×10³ copolymer Copolymer [wt %] [wt %] [wt %] [wt %] [mol/g] precipitatesA 50 50 — — 25 3.8-4.5 B 50 50 25 4.7-5.1 C 30 — 35 35 25 5.0-5.6 D 3070 — — 25 4.8-5.3 E 10 45 — 45 25 4.5-7.0 L-100 50 50 — — 100 3.7-4.3L-100-55 50 — 50 — 250 4.6-5.0 S-100 30 70 — — 100 4.7-5.2

Results:

The (meth)acrylate copolymers, except copolymer E, show relativelynarrow transition ranges of from 0.4 to 0.7 pH units.

Copolymer E appears, because of its low content of 10% by weightmonomers with carboxyl group residues, to have a wider pH transitionrange of 2.5 pH units.

The molecular weight appears to have virtually no effect on thesolubility characteristics of the copolymers.

Example 2 Concentration-Dependent Haemolytic Activity at pH 7.4

The cytotoxicity test with human red blood cells was carried out by amethod based on that of Murthy et al. (N. Murthy, J. R. Robichaud, D. A.Tirrell, P. S. Stayton, and S. Hoffman. The design and synthesis ofpolymers for eukaryotic membrane disruption. J. Controlled Release, 61:137-143 (1999)).

This entails human red blood cells (RBC) being separated bycentrifugation in the presence of K3 EDTA. The cells are in this casesedimented at 200 g and 4° C. for 5 min and subsequently washed threetimes by renewed centrifugation and taking up in phosphate-bufferedsaline (PBS buffer, 34 mM, pH 7.4, 0.9% NaCl weight/volume (75 mM)). Thecell count in the resulting suspension can be determined using ahaemocytometer.

The haemolysis test is carried out by adding the human red blood cells(RBC) in the particular medium to a copolymer suspension at a cellconcentration of 1×10⁸ RBC/ml. The mixture is incubated at 37° C. for 20minutes. The degree of haemolysis was measured by spectrometricdetermination of the haemoglobin released from lysed cells in thecentrifugation supernatant at 541 nm.

The results are shown in Table 2 below. Haemolytic activity [%] at pH7.4 with a Mw × 10³ copolymer concentration in [μg/ml] Copolymer [mol/g]150 250 500 2 500 10 000 A 25 <5 <5 <5 <5 <5 B 25 <5 5 8 25 100 C 25 <5<5 5 100 100 D 25 <5 <5 <5 100 100 E 25 <5 <5 <5 12 20 L-100 100 <5 <5<5 <5 <5 L-100-55 250 <5 <5 <5 <5 30 S-100 100 <5 <5 <5 <5 <5

Result:

Copolymers B and C show a haemolytic activity of 5% or more (toxicitythreshold) at pH 7.4 at 500 μg/ml and upwards. Copolymers B, C, D and Eshow haemolytic activities of more than 5% at 2.500 μg/ml. Thehaemolytic activity of copolymer B is in this case less than that of Cand D.

Copolymers A, L-100, L-100-55 and S-100 have no haemolytic activity evenat high concentrations up to 10 000 μg/ml. Copolymer L-100-55, which isidentical to copolymer B apart from its molecular weight, and coplymer Eshow a slight haemolytic activity at 10 000 μg/ml.

Copolymers A and B have an identical 50% by weight methacrylic acidcontent and differ only in the comonomer—respectively methylmethacrylate and ethyl acrylate. The somewhat more hydrophobic copolymerB is haemolytic even at 250 μg/ml. By contrast, copolymer A is nothaemolytic even at 10 000 μg/ml.

Example 3 Haemolytic Effect of Copolymers B and C in DifferentConcentrations at pH 5.0 and pH 5.5

The results are shown in Table 3 below. TABLE 3 Haemolytic activity [%]at a copolymer concentration in [μg/ml] Copolymer pH 25 50 100 150 250 B5.0 10 80 80 80 80 B 5.5 50 90 95 90 90 L-100-55 5.0 <5 80 80 80 80L-100-55 5.5 5 55 75 90 90 C 5.0 <5 <5 10 60 80 C 5.5 5 50 80 85 85

Result:

Copolymer B is more haemolytic than copolymer C at pH 5.0 and at pH 5.5,especially at low concentrations of 25 and 50 μg/ml. Both copolymers arevery active at pH 5.5 and 150 μg/ml.

Copolymer L-100-55 differs from copolymer B by its higher molecularweight. The haemolytic activity of copolymer B is higher than that ofcopolymer L-100-55 at pH 5.0 and pH 5.5 at 25 and 50 μg/ml as well as atpH 5.5 and 100 μg/ml.

Example 4 Haemolytic Effect at a Copolymer Concentration of 150 μg/mland Various pH Values

The results are shown in Table 4 below. TABLE 4 Haemolytic activity [%]with a copolymer Mw × 10³ concentration of 150 [μg/ml] Copolymer [mol/g]pH 5.5 pH 6.0 pH 6.5 pH 7.0 pH 7.5 A 25 <5 <5 <5 <5 <5 B 25 80 55 <5 <5<5 C 25 90 55 <5 <5 <5 D 25 <5 <5 <5 <5 <5 E 25 <5 <5 <5 <5 <5 L-100 100<5 <5 <5 <5 <5 L-100-55 250 95 80 <5 <5 <5 S-100 100 <5 <5 <5 <5 <5

Result:

Copolymers B, C and L 100-55 show strong haemolytic activity in aconcentration of 150 μg/ml in the pH range from 5.5 to 6.0. All thecopolymers contain ethyl acrylate as comonomer. The Mw of L 100-55 istoo high for applications, however.

Example 5

The intention was to investigate the effect of the copolymers onmacrophage cell types. In contrast to red blood cells, with whichcytolysis presumably takes place mainly through an interaction of thecopolymers with the outer cell membrane, macrophage cell types arecapable of active uptake of the copolymers, so that it must be assumedthat other interactions are able to cause cytolysis or other toxiceffects in this case. Since the determination of cytolysis or othertoxic effects is relatively inexact, two test systems are combined: theMTT assay to access cell proliferation and the LDH assay to accesscellular necrosis (MTT test for surviving cells and the LDH test forkilled cells).

MTT test

MTT test (inhibition of cell proliferation of a macrophage-type cellline).

The calorimetric test detects living cells which reduce the yellow MTTdye to a dark blue formazan product. The test is suitable for detectingcytotoxicity, cell proliferation and cell activation (see, for example,T. Mosmann. Rapid calorimetric assay for cellular growth and survival:Application to proliferation and cytotoxicity assays, J. Immunol. Meth.65: 55-63 (1983)).

J 774 mouse macrophage-type cells are suspended in DMEM mediumcontaining 10% vol/vol foetal calf serum (pretreated at 56° C., 30 min)and 100 U/ml penicillin G and 100 μg/ml streptomycin. The cells aredistributed in 100 μl portions containing 5×10³ cells in a microtiterplate with 96 wells and incubated under an H₂O-saturated atmospherecontaining 5% CO₂ at 37° C. for 24 h. 20 μl of sterile copolymersolution in phosphate buffer are added, and serial dilutions from 0.5 to0.003125 mg/ml are carried out (controls receive phosphate bufferwithout copolymer). Incubation is then continued for 48 h.

10 μg of a sterile MTT solution(MTT=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) inphosphate buffer (5 mg/ml) are put in each well and incubated at 37° C.for a further 3 h. Then 100 μl of a 10% weight/volume sodium dodecylsulphate solution (SDS) in 0.01 M HCl are added in order to solubilizereduced MTT. The absorption at 570 nm is measured in a spectrometerafter 24 h and is evaluated in relation to a 100% survival rate in thecontrol experiment without addition of copolymer.

LDH test

The colorimetric test detects lactate dehydrogenase (LDH) activityreleased by killed cells. The test is carried out in analogy to the MTTtest with J774 cells in microtiter plates. After incubation for 48 h, 4μl portions of the supernatant are tested for LDH activity with acommercially available LDH test kit. Evaluation takes place in relationto 100% cytolysis (toxicity) in the control experiment without additionof copolymer. The 100% value is in this case determined after incubationof a cell aliquot in the presence of Triton X-100 for completecytolysis.

The results are shown in Table 5 below. TABLE 5 MTT test LDH test Co- %cell survival % LDH released pol- 0.03125 0.5 0.03125 0.5 ymer MAA MMAEA MA [mg/ml] [mg/ml] [mg/ml] [mg/ml] A 50 50 — — 90 100 <5 <5 B 50 5030 20 35 40 C 30 — 35 35 100 80 <5 15 D 30 70 — — 100 100 <5 <5 E 10 45— 45 100 <5 <5 40MAA = [wt %] methacrylic acidMMA = [wt %] methyl methacrylateEA = [wt %] ethyl acrylateMA = [wt %] methyl acrylate

Result:

The measurements obtained in the MTT test and LDH test agreequalitatively. Copolymer B is the most toxic for J774 even in lowconcentration. No toxic effect was detectable for copolymers A and D.Copolymer E, which causes limited haemolysis of red blood cells inExample 4, proves to be toxic for J774 cells in a concentration of 0.5mg/ml. Copolymer C proves to be slightly toxic for the J774 cells in aconcentration of 0.5 mg/ml.

Example 6 pH-Dependent Conformational Change (transition) of CopolymersA to E

Pyrene fluorescence

The fluorescent dye pyrene can be used to follow the transition ofpH-sensitive polymers at different pH values (see, for example: K.Kalyanasundaram and J. K. Thomas. Environmental effects on vibronic bandintensities in pyrene monomer fluorescence and their application instudies of micellar systems. J. Am. Chem. Soc. 99:2039-2044 (1977)).

The transitions from the hydrophilic coiled structures (coil) tohydrophobic globule structures are associated with a decrease in theratio of the first (372 nm) and the third (383 nm) peaks the emissionspectrum (I₁/I₃ ratio). For the measurement, pyrene is simply added tothe copolymer solution.

The results are shown in Table 6 below. TABLE 6 Co- Pyrene emission pol-(I₃₇₂ nm/I₃₈₃ nm) ymer MAA MMA EA MA pH 5.5 pH 6.0 pH 6.5 pH 7.4 A 50 50— — 1.32 1.39 1.45 1.52 B 50 50 1.25 1.39 1.46 1.48 C 30 — 35 35 1.271.31 1.35 1.46 D 30 70 — — 1.22 1.23 1.27 1.36 E 10 45 — 45 1.27 1.261.26 1.26

Result:

Copolymers A and B (50% by weight methacrylic acid) show the mostpronounced transition from pH 7.4 to pH 5.5, followed by copolymers Cand D (30% by weight methacrylic acid). Copolymer E (10% by weightmethacrylic acid) shows virtually no transition behaviour. Thetransition behaviour appears to correlate with the methacrylic acidcontent of the copolymers but not with the haemolytic activity of thecopolymers (see Examples 2 and 4).

Example 7 Coupling of Copolymer B with the Dye Lissamine® (Rhodamine B)

The test is carried out as described in K. Abdellaoui, M. Boustta, M.Vert, H. Morjani, M. Manfait. (1998) Eur J Pharm Sci 6, 61-73, with thefollowing modifications. The carboxyl group-activatorN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was replaced by1,3-diisopropylcarbodiimide (DIPC). 4-(Dimethylamino)pyridine (DMAP) andtriethylamine are added in catalytic amounts. The reaction is carriedout for 4 days.

250 mg of copolymer B were dissolved in 1 ml of previously distilledtetrahydrofuran (THF). Then 0.5 mg/ml (3×10⁻³ mmol)1,3-diisopropylcarbodiimide (DIPC) were added to activate the carboxylgroups. After 30 min, catalytic amounts of triethylamine and4-(dimethylamino)pyridine and Lissamine® rhodamine B ethylenediamine (1mg/ml, 1.5×10⁻³ mmol), dissolved in anhydrous N,N-dimethylformamide. Thereaction mixture was incubated at room temperature in the dark underargon for 96 h.

The reaction mixture was passed through a 0.2 μm filter to remove theprecipitated DIPC by product. The THF present was stripped off in vacuo.To the crude product still containing THF a mixture ofchloroform/methanol (85/15 vol/vol) was added and the solution waseluted on a silica gel column to eliminate free rhodamine. The elutionprocess in this case was monitored by thin-layer chromatography. Theappropriate fractions were combined and the solvent was stripped off invacuo. The residue was taken up in THF and dialysed againstwater/methanol (50/50) using a 3 500 dalton membrane for 3 days in orderto remove residues of noncovalently bound rhodamine B. The dialysis wasthen continued against phosphate buffer pH 9 for 2 days and againstdistilled water for a further 2 days. The resulting product was freezedried for 72 h.

The rhodamine B content in the copolymer was determined byspectrofluorometry in methanol at room temperature (λ_(exc)=560 nm,λ_(em)=580 nm).

The yield of crimson-coloured copolymer was 83%. The, rhodamine Bbinding was found to be 0.04 mol %, which means that theoretically onein 9 copolymer molecules is coupled to a rhodamine molecule.

It was intended to test the solubility characteristics at various, pHvalues, comparing with copolymer not coupled to rhodamine B.

The results are shown in Table 7 below. TABLE 7 Insolubility(precipitation) in [%] at pH pH 4.2 4.5 5.3 5.6 6.0 7.0 Copolymer B 10070 5 <5 <5 <5 Copolymer B 70 70 100 10 <5 <5 rhodamine B

Result:

Rhodamine coupling to copolymer B brings about a marked change in thesolubility characteristics. There is a steep rise from about pH 5.6 to100% insolubility at pH 5.3. In contrast to this, the uncoupledcopolymer B does not show 100% insolubility until the pH is 4.2. Therise in this region takes place distinctly less steeply from about pH5.3.

Example 8

Preparation processes: one example is given for each of preparationprocesses 1 and 2. The proportionate amounts can be varied as indicatedin the table on page 38.

The weight average molecular weight (Mw) can be calculated approximatelyfrom the measurements for the viscosity n_(spec/c) (cm³/g) relative tothe viscosity of polymethyl methacrylate (PMMA) at 25° C. in DMF(dimethylformamide). The following empirically found formula is used forthis purpose:$M = ( \frac{\lbrack\eta\rbrack}{4.976 \cdot 10^{- 3}} )^{\frac{1}{0.796}}$

Preparation process 1

1 430 g of distilled water, 3.78 g of sodium lauryl sulphate, 12.6 g ofpolyoxyethylene 20 sorbitan monooleate and 1.26 g of ammoniumperoxodisulphate, dissolved in 20 g of distilled water, were introducedinto a reaction vessel with a capacity of 2 l, equipped with refluxcondenser, stirrer and feed vessel. At 81° C., a monomer mixtureconsisting of:

-   -   270 g of ethyl acrylate    -   270 g of methacrylic acid    -   27 g of 2-ethylhexyl thioglycolate        was metered into a solution over the course of 2.5 hours.

After the feed was complete, the mixture was kept at 81° C. for afurther 2 hours, a mixture of 0.176 g of SE-2MC silicone antifoamemulsion and 10 g of distilled water was added, and 95.42 g of distilledwater was stripped off in vacuo at about 300 mbar, and cooled to roomtemperature. The dispersion has a solids content of 30%.

Preparation process 2

774 g of distilled water, 1.092 g of sodium lauryl sulphate and 1.4 g ofsodium peroxodisulphate, dissolved in 10 g of distilled water, wereintroduced into a reaction vessel with a capacity of 2 l, equipped withreflux condenser, stirrer and feed vessel. At 75° C., an emulsionconsisting of:

-   -   390 g of methyl acrylate    -   150 g of methyl methacrylate    -   60 g of methacrylic acid    -   1.008 g of sodium lauryl sulphate    -   7 g of polyoxyethylene 20 sorbitan monooleate    -   30 g of 2-ethylhexyl thioglycolate    -   820.99 g of distilled water        was metered into this solution over the course of 4 hours.

After the feed was complete, the mixture was kept at 75° C. for afurther 2 hours, a mixture of 0.21 g of SE-2MC silicone antifoamemulsion and 10 g of distilled water was added, and 120 g of distilledwater were stripped off under about 300 mbar, and cooled to roomtemperature. The dispersion has a solids content of 30%. Polym. SolidsBatch Polymer composition Regulator Preparation temp. EmulsifierInitiator content n_(spec/c) calc. No. (% by wt) (parts) process (° C.)(parts) (parts) (%) (cm³/g) Mw****  1 A-B-C = 65-25-10 5 G 2 75 0.15 I0.1 L 30 10 14114 0.5 K  2* B-C = 51-49 3 G 2 80 0.125 I 0.125 J 40 17.728918  3 D-C = 50-50 5 G 1 81 0.3 I 0.1 J 30 13 19624 1 K  4** B-C =70.7-29.3 5 G 2 80 0.125 I 0.13 J 40 10.2 14469  5 A-D-C = 40-30-30 5 G2 75 0.15 I 0.1 L 30 11.7 17191  6 D-C = 50-50 5 G 1 81 0.3 I 0.1 J 3013.7 20961 1 K  7 D-E-C = 49.5-0.5-50 5 G 1 81 0.3 I 0.1 J 30 13.9 213461 K  8 D-E-C = 49-1-50 5 G 1 81 0.3 I 0.1 J 30 13.9 21346 1 K  9 D-E-C =49.5-0.5-50 10 H 1 81 0.3 I 0.1 J 30 12.3 18306 1 K 10 D-E-C = 49-1-5010 H 1 81 0.3 I 0.1 J 30 12 17747 1 K 11 D-F-C = 40-10-50 10 H 1 81 0.3I 0.1 J 30 12 17747 1 K *Final polymerization with 0.01 part of sodiumdisuiphite, 0.0007 part of iron(II) sulphate, 0.05 part of ammoniumperoxodisulphate **Final polymerization with 0.01 part of sodiumdisuiphite, 0.0007 part of iron(II) sulphate, 0.055 part of ammoniumperoxodisulphate ***calculated molecular weight (Mw) relative to PMMA at25° C./DMF$M = ( \frac{\lbrack\eta\rbrack}{4.976 \cdot 10^{- 3}} )^{\frac{1}{0.796}}$A = methyl acrylate B = methyl methacrylate C = methacrylic acid D =ethyl acrylate E = dodecyl methacrylate F = butyl methacrylate G =2-ethylhexyl thioglycolate H = dodecyl mercaptan I = sodium laurylsulphate J = ammonium peroxodisulphate K = polyoxyethylene 20 sorbitanmonooleate L = sodium peroxodisulphate

1. A pH-sensitive polymer comprising 20 to 65% by weight of methacrylicacid units and 80 to 35% by weight of units of C₁- to C₁₈-alkyl estersof (meth)acrylic acid, wherein the pH-sensitive polymer has a molecularweight in the range from 1 000 to 50 000 g/mol, and brings about atleast 60% haemolysis at pH 5.5, and less than 5% haemolysis at pH 7.4,at a concentration of 150 μg/ml in a cytotoxicity test with human redblood cells.
 2. The pH-sensitive polymer according to claim 1, whereinthe pH-sensitive polymer comprises 40 to 60% by weight of methacrylicacid units and 60 to 40% by weight of ethyl acrylate units.
 3. ThepH-sensitive polymer according to claim 1, wherein the pH-sensitivepolymer comprises 20 to 40% by weight of methacrylic acid units, 25 to45% by weight of methyl acrylate units, and 25 to 45% by weight of ethylacrylate units.
 4. The pH-sensitive polymer according to claim 1,wherein the pH-sensitive polymer comprises 40 to 60% by weight ofmethacrylic acid units, 60 to 30% by weight of ethyl acrylate units and2 to 20% by weight of butyl methacrylate.
 5. The pH-sensitive polymeraccording to claim 1, wherein the pH-sensitive polymer comprises 40 to60% by weight of methacrylic acid units, 60 to 40% by weight of ethylacrylate units and 0.1 to 2% by weight of units of a C₈- to C₁₆-alkylester of acrylic or methacrylic acid.
 6. The pH-sensitive polymeraccording to claim 1, wherein at a concentration of 0.03125 mg/ml thepH-sensitive polymer brings about in the MTT test with the mousemacrophase-like cell type J774A.1 (ATCC TIB-67) a percentage-value ofcell survival of at least 25%, based on a 100% survival rate in thecontrol experiment.
 7. The pH-sensitive polymer according to claim 1,wherein at a concentration of 0.03125 mg/ml the pH-sensitive polymerbrings about in the LDH test with the mouse macrophage-like cell typeJ774A.1 (ATCC TIB-67) a LDH release-value of at not more than 40%, basedon 100% cytolysis (toxicity) in the control experiment.
 8. ThepH-sensitive polymer according to claim 1, wherein the pH-sensitivepolymer is in the form of a conjugate or a complex with apharmaceutically effective natural or synthetic biomolecule or an activepharmaceutical ingredient.
 9. The pH-sensitive polymer according toclaim 1, wherein the pH-sensitive polymer is coupled to aconformation-altering agent.
 10. The pH-sensitive polymer according toclaim 1, wherein the pH-sensitive polymer is a constituent of a complexcrosslinked via nucleic acids after chemical modification.
 11. A processfor preparing a pH-sensitive polymer according to claim 1, the processcomprising: free-radically polymerizing 20 to 65% by weight ofmethacrylic acid monomer units with 80 to 35% by weight of monomer unitsof C₁- to C₁₈-alkyl esters of (meth)acrylic acid in the presence ofpolymerization initiators and molecular weight regulators by blockpolymerization, bead polymerization, emulsion polymerization, grouptransfer polymerization (GTP), or atom transfer radical polymerization(ATRP) to form the polymer, discharging the polymer, dissolving thepolymer, purifying the polymer and drying the polymer.
 12. The processaccording to claim 11, wherein the molecular weight regulator is dodecylmercaptan and/or 2-ethylhexyl thioglycolate.
 13. A medicinal substancecomprising the pH-sensitive polymer according to claim 1 as a carrierfor biomolecules or active pharmaceutical ingredients, a conjugate forbiomolecules or active pharmaceutical ingredients, a complex forbiomolecules or active pharmaceutical ingredients, or as a constituentof microparticles, nanoparticles, liposomes, emulsions and/or lipidvesicles.
 14. The medicinal substance according to claim 13 wherein saidbiomolecules are selected from the group consisting of lipids, proteins,peptides, nucleic acids and mixtures thereof.
 15. The medicinalsubstance according to claim 13 wherein the active pharmaceuticalingredients are selected from the the group consisting of analgesics,antiallergics, antirheumatics, antibiotics, antiinfectives,antiparkinson agents, antipsoriatics, antitumour agents,dermatologicals, gout remedies, immunoregulators, gastrointestinalagents, neurotropic agents, opthalmologicals, cytostatics and mixturesthereof.
 16. The medicinal substance according to claim 13, wherein saidmedicinal substance is in a dermal, transdermal, parenteral, nasal,pulmonary, vaginal or oral dosage form.
 17. The medicinal substanceaccording to claim 16 wherein said medicinal substance is effective intreating a disease selected from the group consisting of cancer,infections, cardiovascular disorders, arthritis, neurodegenerativedisorders, genetically related enzyme-deficiency disorders, hepatitis Band C, mucoviscidosis, hypercholesteraemia, Down's syndrome, musculardystrophy, autoimmune diseases, shingles and herpes, psoriasis, CMVretinitis, Crohn's disease, ulcerative colitis, diabetes and mixturesthereof.
 18. The medicinal substance according to claim 13 wherein saidbiomolecules are selected from the group consisting of oligonucleotides,nucleosides, antisense DNA, antisense RNA, nucleotides, toxins,immunotoxins, antibodies, fragments of antibodies and mixtures thereof.